Mechanism of histamine-induced calcium efflux from cultured bovine adrenal chromaffin cells: possible involvement of an Na/Ca exchange mechanism

The effect of stimulation of the histamine receptor on Ca²⁺ mobilization in cultured bovine adrenal chromaffin cells was examined. Histamine (10⁻⁵ M) increased the intracellular free Ca²⁺ ([Ca²⁺]_i) to a peak in the presence or absence of extracellular Ca²⁺, followed by decrease with time. Histamine (10⁻⁸–10⁻⁵ M) also stimulated ⁴⁵Ca²⁺ efflux from cultured bovine adrenal chromaffin cells in a concentration dependent manner. Its stimulatory effect on ⁴⁵Ca²⁺ efflux was inhibited by the specific histamine H₁ receptor antagonist mepyramine. The increase in histamine-stimulated ⁴⁵Ca²⁺ efflux was inhibited by deprivation of extracellular Na⁺ and by the Na⁺/Ca²⁺ exchange inhibitor amiloride. In addition, histamine stimulated ²²Na⁺ influx into the cells, and this action was inhibited by amiloride. These results suggest that stimulation of the histamine H₁ receptor regulates Na⁺/Ca²⁺ exchange in cultured bovine adrenal chromaffin cells.     

Lack of effect of opioid compounds on angiotensin II responses of bovine adrenal medullary cells

Angiotensin II (10 nM) increased basal adrenaline and noradrenaline secretion from cultured bovine adrenal chromaffin cells by 2.5- to 3-fold and 4- to 6-fold, respectively, and stimulated basal accumulation of inositol phosphates more than 2-fold. Etorphine and diprenorphine in the range 10⁻⁹ to 10⁻⁵ M had no effect on the catecholamine secretion induced by angiotensin II, and, at 10⁻⁸ and 10⁻⁵ M, had no effect on angiotensin II-induced inositol phosphate accumulation. The functions of adrenal medullary opioid receptors remain to be determined.     

Modulation of the dihydropyridine-insensitive Ca influx by 8-bromo-guanosine-3′:5′-monophosphate, cyclic (8-Br-cGMP) in bovine adrenal chromaffin cells

Pretreatment of chromaffin cells with the permeable analogue of cGMP, 8-Br-cGMP (100 μM), leads to a reduction (35%) of depolarization-evoked intracellular calcium concentration ([Ca²⁺]_i) increases. There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca²⁺ influx pathways. Combined incubations with nifedipine 10 μM and 8-Br-cGMP reduced KCl-evoked intracellular Ca²⁺ concentration to a greater extent that each compound separately. Moreover, 8-Br-cGMP failed to affect the [Ca²⁺]_i transient induced by the L-type Ca²⁺ channel agonist Bay K 8644 (1μM) under conditions of low depolarization. Neomycin (0.2 mM) and θ-Aga Toxin-IVA (AgTx) (1μM) inhibited the calcium transient to a similar extent, and this inhibition was not enhanced by the presence of 8-Br-cGMP. It is concluded that 8-Br-cGMP modulated the dihydropyridine-insensitive Ca²⁺ influx pathway in the chromaffin cell.     

Improvement in the basic technology of electrofusion for generation of antibody-producing hybridomas

In order to define the optimum conditions of electrofusion technique for the generation of antibody-producing hybridomas, mouse spleen cells or EBV-transformed human B cells were fused with mouse myeloma cells (SP2/0) or human fusion partner cells (KR-4 or KR-12), respectively, by electric field pulse under various conditions. The results confirm reports that the presence of both Ca²⁺ and Mg²⁺ in fusion medium and pretreatment of mixed cells with proteases improved hybridoma yield. Moreover, the presence of liposome or hydrophobic protein in the fusion medium greatly enhanced the yield. Under optimum conditions, hybridoma yields of mouse cells and human cells were 2.5 × 10⁻⁴ and 1 ×10⁻⁴, respectively. These efficiencies were about ten times higher than those obtained by the conventional polyethylene glycol technique. Microscopic observation of the fusion-process revealed that in a human cell system 20%–50% of the cells were physically fused, although only one in 5000 physically fused human cells grew as a hybridoma after hypoxanthine-aminopterin-thymidine selection.     

Spatial and dynamic changes in intracellular Ca measured by confocal laser-scanning microscopy in bullfrog sympathetic ganglion cells

Confocal laser scanning microscopy (CLSM) was used to record spatial and dynamic changes in the intracellular Ca²⁺ ([Ca²⁺]_i) of bullfrog sympathetic ganglion cells in excised tissue or in culture. A CLSM utilizing Ar ion laser (488 nm) and recording fluo-3 fluorescence yielded the sliced image of ganglion cells, while conventional epifluorescence microscopy provided the cell image of a convex structure. A high K⁺ (50 mM) solution, caffeine (3–10 mM) and electrical stimulation (10–20 Hz, 0.5–10 s) caused a homogeneous increase in fluo-3 fluorescence with or without regional differences, possibly due to intracellular organelles and other constituents. Scanning a single horizontal line across the cytoplasm with He-Cd laser (325 nm) and recording indo-1 fluorescence demonstrated that the rate of rise in [Ca²⁺]_i following action potentials depends on the distance from the cell membrane and on the cytoplasmic constituents, showing an inward spread of ‘Ca²⁺-wave’ at variable speeds of 17–219 μm/s. These results suggest that heterogeneity of the cytoplasmic structures and constituents affects dynamic and spatial changes of [Ca²⁺]_i in response to stimuli in neurones. Such heterogenic changes in [Ca²⁺]_i would better be studied by CLMS.     

The role of Ca2+ in the generation of spontaneous astrocytic Ca2+ oscillations

Astrocytes in the rat thalamus display spontaneous [Ca²⁺]_i oscillations that are due to intracellular release, but are not dependent on neuronal activity. In this study we have investigated the mechanisms involved in these spontaneous [Ca²⁺]_i oscillations using slices loaded with Fluo-4 AM (5 μM) and confocal microscopy. Bafilomycin A1 incubation had no effect on the number of spontaneous [Ca²⁺]_i oscillations indicating that they were not dependent on vesicular neurotransmitter release. Oscillations were also unaffected by ryanodine. Phospholipase C (PLC) inhibition decreased the number of astrocytes responding to metabotropic glutamate receptor (mGluR) activation but did not reduce the number of spontaneously active astrocytes, indicating that [Ca²⁺]_i increases are not due to membrane-coupled PLC activation. Spontaneous [Ca²⁺]_i increases were abolished by an IP3 receptor antagonist, whilst the protein kinase C (PKC) inhibitor chelerythrine chloride prolonged their duration, indicating a role for PKC and inositol 1,4,5,-triphosphate receptor activation. BayK8644 increased the number of astrocytes exhibiting [Ca²⁺]_i oscillations, and prolonged the responses to mGluR activation, indicating a possible effect on store-operated Ca²⁺ entry. Increasing [Ca²⁺]_o increased the number of spontaneously active astrocytes and the number of transients exhibited by each astrocyte. Inhibition of the endoplasmic reticulum Ca²⁺ ATPase by cyclopiazonic acid also induced [Ca²⁺]_i transients in astrocytes indicating a role for cytoplasmic Ca²⁺ in the induction of spontaneous oscillations. Incubation with 20 μM Fluo-4 reduced the number of astrocytes exhibiting spontaneous increases.

This study indicates that Ca²⁺ has a role in triggering Ca²⁺ release from an inositol 1,4,5,-triphosphate sensitive store in astrocytes during the generation of spontaneous [Ca²⁺]_i oscillations.     

Effects of noradrenaline on some potassium currents in CA1 neurones in rat hippocampal slices

Pyramidal (CA1) cells in rat hippocampal slices were voltage clamped using a single electrode voltage clamp. In the presence of tetrodotoxin (TTX), depolarizing pulses from holding potentials of −60 to −70 mV elicited a slow inward calcium (Ca²⁺) current and two outward potassium (K⁺) currents: an A current and a slower, Ca²⁺-dependent K⁺ current. Noradrenaline (NA) (20 μM) depressed the amplitude of the K⁺ currents without affecting the Ca²⁺ current. The effect of NA could be blocked with propranolol and was mimicked by isoprenaline, suggesting that NA depresses the K⁺ currents by binding to β-receptors.     

Vip Modulates Intracellular Calcium Oscillations in Human Lymphoblasts

Vasoactive intestinal polypeptide (VIP) has been shown to stimulate adenylate cyclase in a human lymphoblast cell line (MOLT 4). In the present study, we monitored fluorescence in cell suspensions and in single fura-2 loaded MOLT 4 lymphoblasts to determine if VIP modulates intracellular calcium concentrations ([Ca²⁺]_i), and if this modulation is mediated by adenylate cyclase. The distribution of [Ca²⁺]_i in resting and stimulated cells was non-homogeneous, with gradients of high [Ca²⁺]_i present in the subplasmalemmal space. In a subset of cells (10-30% of all cells studied), [Ca²⁺]_i showed La³⁺-sensitive, temporal changes in the form of [Ca²⁺]_i oscillations with a baseline [Ca²⁺]_i value of 115±10 nM, an oscillation amplitude of 150±18 nM and a mean period of 9.2±2s. The remaining non-oscillating cells showed a constant [Ca²⁺]_i level of 75±5 nM (n=65 cells from 4 experiments). In the subset of cells with spontaneous [Ca²⁺]_i oscillations, VIP dose-dependendy (10^-12 to 10^-8M) increased the amplitude of oscillations but did not stimulate their frequency. The stimulatory effect of VIP was correlated with baseline [Ca²⁺]_i in these cells, was attenuated in the presence of La³⁺ (25 μM), but was unaffected by cell depolarization (126 mM KC1). Dibutyryl cyclic AMP (10^-4 to 10^-3 M) and forskolin (10^-4M) had no effect on [Ca²⁺]_i oscillations, or on [Ca²⁺]_i in cells without oscillations. In cell suspensions, baseline [Ca²⁺]_i was found to be 55. 1±11.2 nM (meanS.E.M., n=11); VIP, cyclic AMP analogues or forskolin had no significant effect on [Ca²⁺]_i. These findings suggest that: a) VIP modulates the amplitude of [Ca²⁺]_i oscillations generated by a cytosolic [Ca²⁺] oscillator in a subset of cells at a concentration of 10^-12M, a thousand-fold below the K_D for the VIP receptor; b) baseline [Ca²⁺] values may be related to both the ability of cells to generate spontaneous [Ca²⁺] oscillations and of oscillating cells to respond to VIP; c) due to the small number of responding cells, VIP-induced [Ca²⁺]_i changes are not detectable when studied in cell suspensions.     

Effects of free Ca on the [Ca + Mg-Dependent adenosinetriphosphatase (ATPase) of Alzheimer and normal fibroblasts

Differences between Alzheimer and control fibroblasts [Ca²⁺ + Mg²⁺]-dependent ATPase activity at free Ca²⁺ concentration considerably higher than physiologic concentrations were observed. At 50 μM free Ca²⁺, Alzheimer and control fibroblast homogenates exhibited maximum velocity values ranging from 8 to 25 nmoles phosphate released/min/mg protein. Higher free Ca²⁺ (350 μM) inhibited control fibroblast ATPase activity approximately 77%; whereas, Alzheimer fibroblasts retained greater than 75% starting activity. Although the pathophysiological significance of these findings is at present unclear, these data suggest the Ca²⁺ pump of Alzheimer fibroblasts behaves differently in the presence of high free Ca²⁺. Such behavior may be of potential diagnostic value.     

Acetylcholine receptors in dissociated nucleus basalis of Meynert neurons of the rat

Electrical and pharmacological properties of acetylcholine (ACh)-induced currents in neurons dissociated from the nucleus basalis of Meynert (nBM) of immature (2-week-old) rats were investigated with the whole-cell mode of the patch-clamp technique. At a holding potential (V_H) of −50 mV, ACh (10⁻⁴M) evoked a transient inward current mimicked by nicotine (I_nACh), followed by a sustained outward current mimicked by carbamylcholine (I_mACh). The K_D values were 1.2 × 10⁻⁴ M for I_nACh) and 8.7 × 10⁻⁷ M for I_mACh. The reversal potenial of I_mACh was close to E_K. The I_mACh was determined to be elicited via the M₂ muscarinic receptor, based on the differences in sensitivity to muscarinic antagonists such as pirenzepine and AF-DX-116.     

Dual effect of verapamil on K-evoked release of endogenous dopamine from arcuate nucleus-median eminence complex

The effect of verapamil, a calcium-entrance blocker, on K⁺-evoked release of endogenous dopamine from tuberoinfundibular neurons incubated in vitro was studied. This compound, added to the incubation medium, at the dose of 10⁻⁶ M, significantly reinforced K⁺-induced dopamine release, whereas, at higher doses (10⁻⁵, 5 × 10⁻⁵ and 10⁻⁴ M), it completely prevented the stimulated dopamine release. The results obtained with the higher doses showed the calcium dependence of K⁺-evoked release of endogenous dopamine from central neurons. The opposite effect, seen with the lower dose of verapamil, could be due to different pharmacological properties of the drug.     

Adenosine triphosphate mobilizes cytosolic calcium and modulates ionic currents in mouse taste receptor cells

By using photometry and the patch clamp technique, we identified P_2Y-like receptors in mouse taste receptor cells (TRCs) and found them to be coupled to Ca²⁺ mobilization and ionic current modulation. Particularly, adenosine triphosphate (ATP) and the P_2Y agonist 2-methylthio-ATP increased intracellular Ca²⁺ by stimulating the phosphoinositide pathway, whereas β,γ-methylene- -ATP, a P_2X agonist, was ineffective. In a distinctive TRC subpopulation, ATP closed Ca²⁺ channels. This regulation may underlie the negative feedback tuning neurotransmitter release. By mobilizing intracellular Ca²⁺, ATP activated Ca²⁺-dependent Cl⁻ channels, the intracellular event that may universally occur upon taste stimulation triggering IP₃ formation and Ca²⁺ release in the TRC cytoplasm.     

Glutamine-induced membrane currents in cultured chick spinal cord neurons

The effects of -glutamine (GLN) on cultured spinal cord neurons from the chick were studied in the whole cell mode of the patch clamp technique. GLN induced membrane currents rectified at positive membrane potentials (m.p.) and reversed polarity close to zero m.p. The dose-response curve was nearly linear at a semilogarithmic scale for concentrations of 10⁻⁵ M-10⁻² M. Summation of the responses evoked by GLN (10⁻³ M) and glycine (10⁻³ M) was observed when these two amino acids were applied together, while no significant increase of the responses was present when GLN was applied together with -glutamate (10⁻³ M) or kainate (10⁻³ M). It is suggested that GLN binds to the glutamate receptors and activates the same type of ionic channels as glutamate and kainate.     

In vitro effects of adrenomedullin and calcitonin gene related peptide on the release of serotonin and amino acids from rat dorsal spinal cord

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) are structurally related, interact with each others receptors and show overlapping biological activities. Immunoreactivity (IR) and mRNAs along with binding sites for both CGRP and adrenomedullin have been shown in the rat spinal cord. CGRP mediates the transmission of nociceptive information at the spinal cord level and both peptides has shown to induces c-fos expression and accumulation of cAMP in spinal cells. In this study, HPLC methods were used to investigate the effects of AM and CGRP on the basal and K⁺-evoked release of serotonin, glutamate (Glu), aspartate (Asp), glycine (Gly) and γ amino butyric acid (GABA) from the slices prepared from the rat spinal cord. Neither CGRP (10⁻⁷ and 10⁻⁶ M) nor AM (10⁻⁷ and 10⁻⁶ M) had significant effects on the basal release of serotonin and the amino acids tested in this study. However, CGRP produced statistically significant increases in the K⁺-evoked release of Asp and Glu, whereas AM failed to do so. Neither AM nor CGRP (10⁻⁷ and 10⁻⁶ M) showed any significant effects on the K⁺-evoked release of serotonin, GABA and Gly. Present data suggest that the stimulatory effects of CGRP on the release of Asp and Glu were exerted by distinct types of CGRP receptors.     

Modulation of Human T-Lymphocyte Plasma Membrane Ca +2 Permeability by Imidazole Antimycotics

The role of cytochrome P-450 in the regulation of plasma membrane Ca⁺² permeability of human peripheral T-lymphocytes by intracellular Ca⁺² was examined. We assessed the effect of imidazole inhibitors of cytochrome P-450 on the intracytoplasmic free Ca⁺² ([Ca⁺²]i) response generated using the microsomal ATPase inhibitor thapsigargin (THG) to deplete the intracellular Ca⁺² stores. Econazole, miconazole and clotrimazole dramatically inhibited the THG mediated increase in [Ca⁺²]i and indud an increase in [Ca⁺²]i themselves. This inhibitory effect was previously observed in other cell systems and was attributed to inhibition of cytochrome P-450 by these agents. However, we evaluated a variety of structurally dissimilar P-450 inhibitors and found that none affected [Ca⁺²]i, indicating that the mechanism of imidazole action does not involve P-450.     

Characterization of CD28CD4 T cells in living kidney transplant patients with long-term allograft acceptance

CD28⁻CD4⁺ T-cell subpopulation is expanded in kidney allograft patients with long graft survival. To seek for the roles of CD28⁻CD4⁺ T cells in the long-term acceptance of kidney allografts, we characterized this population by analyzing cell surface molecules, TCR V_β repertoire, mixed lymphocyte reaction (MLR), and cytokine production. The number of CD28⁻CD4⁺ T cells increased correlatively with time after transplantation in this group of patients. The CD28⁻CD4⁺ T cells did not express detectable levels of CD25, CD69, V24, or CTLA-4 but expressed heterogeneous amounts of CD45 RA on the surface. Freshly sorted CD28⁻CD4⁺ T cells revealed a restricted V_β repertoire, whereas the V_β usage of CD28⁺CD4⁺ T cells from the same patients was much diversified. Expression levels of TGF-β and IFNγ gene were significantly higher in the CD28⁻ CD4⁺ T cells than in the CD28⁺CD4⁺ T cells from the kidney allograft patients. These findings suggest that an oligoclonal CD28⁻ CD4⁺ T-cell population is continuously activated in patients with long allograft survival, which may be linked with the long-term acceptance.     

Brain oxidation is an initial process in sleep induction

CNS activity is generally coupled to the vigilance state, being primarily active during wakefulness and primarily inactive during deep sleep. During periods of high neuronal activity, a significant volume of oxygen is used to maintain neuronal membrane potentials, which subsequently produces cytotoxic reactive oxygen species (ROS). Glutathione, a major endogenous antioxidant, is an important factor protecting against ROS-mediated neuronal degeneration. Glutathione has also been proposed to be a sleep-promoting substance, yet the relationship between sleep and cerebral oxidation remains unclear. Here we report that i.c.v. infusion of the organic peroxide t-butyl-hydroperoxide at a concentration below that triggering neurodegeneration (0.1 μmol/100 μl/10 h) promotes sleep in rats. Also, microinjection (2 nmol, 2 μl) or microdialysis (100 μM, 20 min) oft-butyl-hydroperoxide into the preoptic/anterior hypothalamus (POAH) induces the release of the sleep-inducing neuromodulators, nitric oxide and adenosine, without causing neurodegeneration. Nitric oxide and adenosine release was inhibited by co-dialysis of the N-methyl-d-aspartate receptor antagonist, d(−)-2-amino-5-phosphonopentanoic acid (D-AP5; 1 mM), suggesting that glutamate-induced neuronal excitation mediates the peroxide-induced release of nitric oxide and adenosine. Indeed, Ca²⁺ release from mitochondria and delayed-onset Ca²⁺ influx via N-methyl-d-aspartate receptors was visualized during peroxide exposure using Ca²⁺ indicator proteins (YC-2.1 and mitochondrial-targeted Pericam) expressed in organotypic cultures of the POAH. In the in vitro models, t-butyl-hydroperoxide (50 μM) causes dendritic swelling followed by the intracellular Ca²⁺ mobilization, and D-AP5 (100 μM) or glutathione (500 μM) inhibited t-butyl-hydroperoxide-induced intracellular Ca²⁺ mobilization and protected POAH neurons from oxidative stress.

These data suggest that low-level subcortical oxidation under the control of an antioxidant system may trigger sleep via the Ca²⁺-dependent release of sleep-inducing neuromodulators in the POAH, and thus we propose that a moderate increase of ROS during wakefulness in the neuronal circuits regulating sleep may be an initial trigger in sleep induction.     

Requirement of neural activity for the maintenance of dopaminergic neurons in rat midbrain slice cultures

Chronic treatment of organotypic midbrain slice cultures with L-type Ca²⁺ channel blocker nicardipine (3–10 μM) or verapamil (10 μM) for 18 days resulted in a drastic decrease in the number of dopaminergic neurons. A voltage-dependent Na⁺ channel blocker tetrodotoxin (1 μM) was also effective in decreasing the number of dopaminergic neurons. Concurrent application of forskolin (20 μM) or dibutyryl cyclic AMP (1 mM) counteracted the effects of nicardipine and tetrodotoxin. These results suggest that spontaneous neuronal activity within midbrain slice cultures, causing Ca²⁺ influx through L-type Ca²⁺ channels that maintains intracellular cyclic AMP levels, is required for the maintenance of dopaminergic neurons.     

Human suppressor T cells: Induction, differentiation, and regulatory functions

T-cell-mediated suppression of human immune responses involves a complex interaction between distinct lymphocyte subsets with suppressor-inducer and suppressor-effector functions. Recent studies with subset-specific monoclonal antibodies have defined a characteristic phenotype of suppressor-inducer cells (CD4⁺ Leu8⁺ 2H4⁺ 4B4⁻) that can be distinguished from that of helper cells for antibody synthesis (CD4⁺ Leu8⁻ 2H4⁻ 4B4⁺). Similarly, suppressor-effector cells (CD8⁺ CD11⁺ Tp44⁻ can typically be defined as a subset separable from cytotoxic T cells (CD8⁺ CD11⁻ Tp44⁺). Both antigen-specific and nonspecific interactions are important in suppressor T-cell activation and function. Soluble signals required for differentiation of CD8⁺ suppressor cells include an indomethacin-sensitive monocyte product and interferon gamma. In contrast, proliferation of the CD8⁺ suppressor cell subset depends on stimulations first by a product of CD4⁺ Leu8⁺ cells, T suppressor cell growth factor, and second by interleukin 2. Although the molecular basis of antigen-specific interactions between CD4⁺ and CD8⁺ cells in suppressor cell generation has not been defined, it may involve both conventional, presumably MHC-restricted, interactions between antigen and antigen receptors, as well as anti-idiotypic interactions of suppressor-effectors with determinants on suppressor-inducer receptors. Progress in elucidating requirements for activation, growth, and differentiation of suppressor cells should facilitate long-term culture of such cells and lead to clearer understanding of mechanism of suppressor-cell mediated immunoregulation.